1. Field of the Invention
The invention generally relates to methods for the treatment of cancer and other neoplastic diseases and, more specifically, to recombinant herpesviruses comprising DNA encoding various cytokines and their use in treating cancer and other neoplastic diseases of the central nervous system.
2. Related Technology
Neoplastic diseases of the central nervous system (CNS) present a tremendous therapeutic challenge in spite of advances in accepted treatment modalities such as surgery, radiotherapy, and chemotherapy. Survival of patients afflicted with certain types of brain tumors from the time of diagnosis is usually measured in months, while recurrence after treatment is normally associated with a life expectancy measured in weeks. More recent therapies involve the use of genetically engineered viruses and immunotherapy to destroy tumor cells.
Genetically engineered (recombinant) viruses have been studied at length for use as vectors for achieving a number of therapeutic objectives. Such objectives include (1) delivery to cells of normal copies of genes to circumvent the pathologic effect of missing or mutated endogenous genes, 2) selective destruction of cancer cells, and 3) immunization with one or more antigens in order to confer immunity to an infectious agent or to stimulate the host's immune system function so as to recognize, for example, tumor associated antigens.
Approaches to viral therapy of neoplastic disease are twofold. A first approach includes the use of non-destructive viruses (e.g. genetically-altered retroviruses) to introduce into cells genes that express an enzyme such as the herpes simplex virus (HSV) thymidine kinase enzyme that the cells do not otherwise express. The rationale of this type of therapy is to selectively provide tumor cells with an enzymatic activity that is lacking or is much lower in the normal cells and which renders the tumor cells sensitive to certain drugs. For example, drugs such as gancyclovir and acyclovir require phosphorylation by thymidine kinase before they are active. Providing tumor cells with the viral thymidine kinase gene allows the cells to phosphorylate and thus activate the drug which may then be incorporated into the DNA of dividing tumor cells or otherwise inhibit DNA synthesis in those cells, thereby leading to the destruction of the cells.
Another approach to viral therapy of neoplastic disease involves direct inoculation of tumor with debilitated or attenuated viruses, which require for replication, certain factors that are present in tumor cells but that are not present in normal cells. For example, tk− HSV mutants (i.e., HSV mutants lacking the tk or thymidine kinase gene) have been directly injected into tumors in mice having CNS neoplastic disease, thereby leading to the infection and ultimate destruction of the tumor cells. Nonetheless, some of the test animals died presumably of viral encephalitis before any tumor-related deaths in the control group. [Martuza et al., Science 252:854-856 (1991)]
The rationale for the use of tk− viruses is based on the fact that such mutant viruses are totally dependent on cellular thymidylate synthetase as a source of thymidine triphosphate for DNA replication. Therefore, these mutant viruses exhibit a reduced virulence for normal central nervous system tissues yet are able to actively multiply in and infect tumor cells which have sufficient levels of thymidylate synthetase to support viral DNA synthesis thereby causing the destruction of the tumor cells.
In practice, a number of limitations to this approach to viral therapy of neoplastic disease exist. Specifically, a major limitation to the use of tk− viruses is that these mutant viruses are not completely avirulent. Another limitation to this approach is the lack of a secondary or alternative mechanism of action. More particularly, in the event complications arise to compromise the primary mechanism of action (i.e., infection and destruction of tumor cells), unlike the tk+ viruses discussed above, the tk− viruses would be unable to phosphorylate such pro-drugs as acyclovir or gancyclovir to an active form (due to the lack of thymidine kinase), thereby not providing or alternative or a secondary mechanism of action.
An alternative to the viral mutants referenced to above is the use of HSV mutants (in which a particular gene or genes are rendered incapable of producing an active gene product) that are unable to grow in the normal CNS cells but which are capable of growth in CNS tumor cells. One such gene is the γ134.5 gene. The γ134.5 gene maps within the inverted repeats ab and b′a′ sequences flanking the unique long (UL) domain of the HSV genome and is, therefore, present in two copies per genome. [Chou et al., Science, 250:1262-1266 (1990).]
Numerous studies have been conducted with HSVs in which the γ134.5 gene or genes have been inactivated by substitutions, deletions, or insertions of mutations. For example, it has been shown that γ134.5 (null) mutants are highly attenuated (PFU/LD50ratios>106) in mice. Further, it has been demonstrated that in cells of human derivation infected with γ134.5− viruses, initiation of viral DNA synthesis induces a total shutoff of protein synthesis and results in reduced viral yields.
Studies using the γ134.5 deletion mutant (R3616) for the therapy of central nervous system tumors indicate that this virus is superior to deletion mutants used previously. More particularly, it was shown that a γ134.5− virus (i.e., the R4009 virus, containing a mutation via insertion of an in-frame stop codon in the γ134.5 genes), is significantly better than the R3616 null mutant in its ability to destroy cancer cells and prolong the life of mice bearing central nervous tumors. In some instances, mice survived tumor free.
The use of genetically engineered herpes simplex virus (HSV) for treatment of malignant gliomas has been described previously. As these studies used immunocompromised mice, a central question arose as to (1) whether the infection induced an immune response to the tumor cells, and (2) whether the response could be modified by cytokines expressed from genes cloned into the virus.
Enhancement of the immune response to malignant gliomas has recently emerged as a major avenue of potential therapy. This approach is based on the observation that patients with malignant brain tumors are immunosuppressed (i.e., immunosuppression of T-cell functions). Although gliomas are poor antigen-presenting cells in vivo with low expression of MHC class I and II antigens, they also secrete several glioma suppressor factors such as TGF-β2 and prostaglandin E2. Therefore, a major goal of cancer immunotherapy is to stimulate recognition of tumor cells by the host's immune system and to activate tumor antigen-specific cellular immunity.
Direct transfer of cytokine genes in tumor cells has emerged as a powerful immunotherapeutic tool in the new approaches for the management of cancer patients. In experiments with animal models, tumor cells transduced with cytokine or growth factor genes such as interleukin IL-1, IL-2, IL-4, IL-6, IL-7, interferon (IFN-γ), tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony stimulating factor (GM-CSF) have demonstrated in vivo inhibition of tumor growth by stimulating localized inflammatory and/or immune responses. In contrast, cytokines like IL-5 and IL-10 fail to stimulate host immunity and do not kill tumor cells. Transforming growth factor β2 (TGF-β2) has been shown to decrease or inhibit immunogenicity.
The therapeutic efficacy of cytokine therapy in intracerebral neoplastic disease has been tested only recently. More specifically, retrovirus vectors have been utilized primarily to transduce cytokine genes into glioma cells. Initial results from these studies have been mixed, at best. Therefore, there remains a need for a more suitable viral vector with which to introduce therapeutic genes, e.g., cytokine genes, into central nervous system tumors, for the purpose of treating the neoplastic disease. Preferably, such viral vectors (i.e., adenovirus, adeno-associated virus and herpes simplex virus or others) are capable of expressing the foreign gene (i.e., cytokine) and/or are capable of replicating conditionally within the tumor area. A further desirable characteristic would be that the viral vector be highly neurotropic. Such characteristics are expected to produce a more potent cytokine-mediated anti-tumor effect as compared to the cytokine-mediated anti-tumor effect obtained via administration of a retroviral vector.